Silicon (Si) is not found in nature in its elemental (metal) form, although it is the second most abundant element after aluminum (Al) in the Earth's crust. Indeed, it has a very great attraction for oxygen and it forms a tetrahedral structure bound to four oxygen in its stable and most predominant form in water as monosilicic acid Si(OH)4. This weak acid shows four acid functions with a lowest pKa value of 9.8. This implies that at pH 9.8 mono-silicic acid is present for 50% in the un-dissociated acid form and for 50% in the mono-ionic salt form (a silicate ion). The other pKa values are between 11.8 and 13.5. This means that only very strong alkali addition is able to dissociate all four acid groups into four silicate ions starting from silicic acid. At pH values higher than 10.8 silicates are predominantly formed.
Between pH 2 and 8 mono-silicic or ortho-silicic acid is a neutral molecule. It is predominantly uncharged between pH 2 and 3. At concentrations higher than 3 mM it starts to polymerize through a condensation reaction. It is therefore impossible without introduction of special stabilizing agents to synthesize high concentrations (≥4 mM) mono-silicic acid, stable in time at room temperature. During a first condensation reaction between two molecules mono-silicic acid and under liberation of a water molecule di-silicic acid is formed (i.e. the dimer (OH)3Si—O—Si(OH)3). This dimer is the crucial starting molecule for further polymerization reactions. Depending on the Si concentration, the temperature and the presence of other molecules or ions, polymerization proceeds with the formation of trimers, tetramers, and bigger oligomers (linear or cyclic). At higher concentrations bigger linear molecules silicic acid are formed which grow further and start to condense together forming colloidal structures or silica.
All these structures in suspension hydrolyze in time upon dilution and smaller molecules are again formed with consumption of water molecules.
Further polymerization of colloids results in the formation of amorphous silica under formation of precipitates or gel. Only the smallest forms of silicic acid (mono- and di-silicic) are bio-available for organisms (plants, algae, lichens, animals, human, etc.). These molecules are available in soil water, rivers, seas, sources, oceans, etc. The silicon concentration of these bio-available acids in water is limited to low concentrations (<3-5 mM). Especially plants and algae convert these acids into biogenic silica which is very slowly dissolved in water into mono-silicic acid.
During the last decade, evidence has come to exist that besides silicic acid also mono-silicates and mono-silicate complexes show bioavailability characteristics. Nevertheless, it is difficult to prove the bioavailability of these compounds.
Small silicic acid molecules are able to diffuse through all kind of cellular membranes and specific aquaporins (entry channels) or transporter proteins for mono-silicic acid were detected in plants and algae. It is evident that silicate ions (mono- or di-silicate ions) could enter membranes in a similar way. It is chemically difficult to show the difference between small silicic acids and their derived silicate ions. It is also possible that the silicate ions are converted into silicic acid during entry into the membrane or that they enter as a complex via another channel. Present invention starts from the finding that compatible solutes, which have their own specific aquaporins, are able to deliver silicates into the cellular membranes.
It is possible that silicic acid and silicates fulfill different activities in the cell. The cell specificity could be different for the ionic form in different organisms. We know that in plants, animals and human, both kinds of silicon are bio-available and that structural or physiological effects are detectable after administration. Until now most accentuated effects were detected after supplementation of silicic acid.
Silicic acid is the natural bio-available silicon source in water. Silicon in food is present under different forms, mostly as biogenic silicic acid and complexes thereof with macromolecules as proteins and sugars. Silicate complexes and insoluble silicates may also be present. There is no correlation between the silicon content in food and the uptake of silicon in human. The bioavailability of the silicon compound is therefore important. The bioavailability of most compounds except for (mono) silicic acid is not studied.
It has been demonstrated, according to this invention, that supplementation of comparable amounts of osmolyte stabilized soluble silicates is able to show similar effects as silicic acid supplementation, whereas non stabilized silicates are less powerful.
The most common silicon forms in nature are silicic acid (from mono silicic acid to insoluble silica) and silicates. Most silicates are aluminosilicates present in soil minerals and rocks. These structures are stable and only broken down by physical forces (mechanical and biological fractures freeze thawing, etc.) followed by chemical weathering through activity of acids. Mono-silicic acid is formed as result from these chemical or biological reactions together with new silicates and is solubilized in water. Silicates form mainly very complex structures and may contain a mixture of different minerals. Silicates are normally solubilized and dissolved in strong alkaline conditions.
Most silicates are more soluble in water at high pH than silicic acid at pH between 2 and 8.
Silica (highly polymerized silicic acid) solubility in water is generally under 200 ppm. In highly concentrated silica industrial waters, silica is removed to inhibit membrane disturbing precipitation or deposition using reverse osmosis or on exchange techniques. Purified drinking water using these techniques contains therefore less or no silicon.
Silica particles exhibit irregular negative charges on the surface but they are not to be considered as real anions resulting in precipitation of salts. They truly precipitate as silicic acid and are only slowly solubilized. The more silica particles contain water (less cross-linking and more OH present) the easier they are solubilized again. The access of OH− and water is essential for the dissolution of polymerized silicic acid and silicates into mono-silicic acid or mono-silicate.
Silica particles contain highly hydroxylated surfaces attracting and binding macro-molecules, containing OH groups, through hydrogen (O—H) bonds as polysaccharides, proteins, phenols, etc.
Silicates are industrially prepared from silica under strong alkaline conditions through dissociation of the Si—O—Si bond and ionization of the Si—OH acid group resulting in a Si—OM (M is a Metal ion) bond. The solubility of the resulting silicates depends on the concentration of OH ions supplemented and the degree of dissolution of Si—O—Si bonds. The higher the silica concentration the more alkaline is used for complete dissolution and solubilisation resulting in a higher mono- and di-silicate concentration, which is required for bioavailability.
The most interesting soluble silicates are the alkali metal silicates and preferably sodium and potassium silicates. They are synthesized from purified silica and highly soluble under alkaline conditions delivering especially mono- and di-silicates.
Such solutions contain normally a mixture of silicate anions. The building block of all these anions is the tetrahedral anion with a silicon atom in the center and oxygen in the corner of the four-sided pyramid: SiO44−, similar to mono-silicic acid. This is the monomeric silicate anion (mono- or ortho-silicate anion). A hydrogen, potassium or sodium ion is associated with each oxygen. Upon polymerization, tetrahedra are linked with each other via Si—O—Si bonds. The negative charge of the unshared oxygen atoms is balanced by the presence of alkali cations, which are randomly spaced in the interstices of the silicate structure.
Because silicates are produced from silica different structures exist depending on the degree of silica polymerization, the concentration of added alkali hydroxide, and the degree of dissolution. Upon dissolution, solubilized silicates give rise to molecular speciation. The mixture of silicate anions in solution shows a complex of mono-, di-, tri- and higher linear, cyclic and three dimensional silicate anion structures represented by the general formulationxSiO2:M2Owhere M is an alkali metal (lithium, sodium or potassium), with x representing the molar ratio (MR) of silica to metal oxide, defining the number of moles silica per mol alkali metal oxide. The higher the molar ratio, the less alkali metal ions are present in the silica network and the less alkaline the silicates are. For industrial applications, the weight ratio is indicated (WR) and is derived from the MR. For potassium silicate MR=1.56 WR. Potassium silicates are mostly produced with weight ratios ranging from 1.3 to 2.5.
In aqueous alkaline solution, a mixture of monomeric, oligomeric and polymeric silicate ions is formed which are in a dynamic equilibrium. This x ratio influences the distribution of anions. Lower (<2) ratios will result in higher concentration of mono and di-silicates and smaller oligorners while higher (>2.2) ratios will result in more complex structures, larger rings and polymers. The pH values of the concentrated products are usually between 10 and 13. Above pH 11-12 stable solutions of monomeric and polymeric silicate ions, without insoluble amorphous silica, are obtained but the solubility rapidly decreases when the pH is lowered to 9. Below this pH only a very small proportion is present as monomeric silicate anion besides insoluble silicate. Polymers or amorphous silica gels are formed, characterized by the loss of interstitial alkali ions from the three-dimensional network.
Dilution of concentrated silicate solutions in water results in new distributions of silicate structures. The pH of the diluted solution will drop and depending on the silicon concentration insoluble silicates are formed. The pH reduction of the diluted silicate is less than might be expected due to the buffering effect of the silicate and their pK values between 10 and 13. The silicates will slowly precipitate or polymerize.
The addition of acid at diluted samples is necessary to form silicic acid starting from concentrated silicate solutions (>0.1 M Si). The silicate anions are able to complex with OH containing macromolecules above pH 8. Soluble silicates can also react with multivalent cations, which are present in all kinds of water and media, forming the corresponding insoluble metal silicate resulting in a decreased bioavailability due to depletion of these ions.
Following soluble silicates are registered according to EU regulation
Sodium silicates (NaO:xSiO2),
Disodium metasilicate, anhydrous,
Disodium metasilicate, pentahydrate,
Disodium metasilicate monohydrate,
Potassium silicates (K2O:xSiO2),
Lithium silicates (Li2O:XSiO2).
Silicon is not yet fully accepted as an essential element for all living organisms although there is ample evidence for beneficial effects in plants, microorganisms, animal and human.
The beneficial effects are particularly pronounced in plants exposed to abiotic and biotic stresses (Epstein E. (1999) Silicon Annual Review of Plant Physiology and Plant Molecular Biology, 50, 641-664). Epstein and Bloom even modified the definition of an essential element for plants to incorporate silicon (Epstein E. and Bloom A. J. (2005) Mineral Nutrition of Plants Principles and Perspectives; 2nd Edition Sunderland, M A, USA; Sinauer associates). In aboveground parts of the plant silicon concentration varies from 0.1 to 10.0% of dry weight. There are differences in silicon uptake and transport resulting in plant classifications regarding silicon uptake. There are plants showing an active uptake and silicon accumulation (some cyperaceous and graminaceous as rice, wheat, ryegrass and barley), other groups tolerate passive diffusion of silicon (some dicotyledonous as cucumber, melon, strawberry and soybean) while some dicots even exclude silicon (as tomato and bean). Silicon transporters were identified in rice responsible for root and xylem loading. There is little information for Si uptake and transport in the other monocots or dicots. It was shown that both active and passive mechanisms are operating in Si uptake and transport in Si accumulator plants (Epstein E. (2009) Annals of Applied Biology, 155, 155-160; Kvedaras O. L. et al. (2009) Annals of Applied Biology, 155, 177-186; Lang Y. C. et al. (2007) Environmental Pollution, 147, 422-428; Brunings A. M. et al. (2009) Annals of Applied Biology, 155, 161-170).
The authors of the present invention performed several experiments with silicon accumulator plants, silicon diffusion plants and silicon rejecting plants using a foliar spray application every week with strongly acidified silicates, resulting in a foliar application with guaranteed silicic acid (solution containing 0002 mg/ml Si).
In all these trials, there was a clear effect on the growth, production, resistance to infection, color of fruits; shelf life etc. showing that silicon is really taken up as silicic acid in all plants using foliar application. Some scientists reject the foliar application technique and only believe in the root amendment technique.
In general, in literature published experiments with silicon fertilization were conducted using either solid (ex.: calcium silicate and sodium silicate) mixed with the soil, or solubilized silicate solutions (ex.: sodium or potassium silicate) using a soil irrigation or a soil mixing technique with appropriate dilutions in process water (pH<9) containing multivalent minerals and other impurities. Silicon concentrations between 1 and 100 mM are normally used (1 mM Si=28 microgram Si/ml). Foliar application is not frequently used but similar concentrations are applied and this foliar application is believed to show less efficiency in laboratory experiments. During soil fertilization, silicates stay much longer around the roots and dissolve slowly while in the case of foliar applied silicates, the thin water-film containing silicon is quickly dried up.
Most authors claim that mono-silicic acid is the active ingredient instead of silicate. It is difficult or quite impossible to show that a diluted silicate concentrate is completely converted into silicic acid, the confirmed and proven bio-available compound. First, there is a drastic decrease in pH upon dilution generating polymerized silica. Omni present multivalent metal ions result in the formation of insoluble silicates and the lack of sufficient protons in process water pH>6 starting from concentrated Si solutions pH>12 inhibits the complete formation off silicic acid. Complexation with —OH containing natural compounds may also occur.
The present invention, using a mixture of selected osmolytes in highly concentrated soluble silicate solutions, resulted in a new formulation probably protecting the silicate ions from polymerization in their concentrated form and during dilution, showing high biological activity after dilution comparable to silicic acid.
Silicon confers tolerance in plants to various abiotic and biotic stresses. It does not only accumulate as biogenic silica through polymerization in cell walls inhibiting fungal or bacterial invasion, protects xylem fluidity, neutralizes toxic metals, acts against salinity and drought, affects structure, integrity and functions of membranes, inhibits lipid peroxidation, improves nutrient balance, increases shelf life of vegetables and fruits, etc.
During the last two decades there are hundreds of publications showing the benefits of silicon in plants, animals and human (Robberecht H. et al. (2009). Science of the Total Environment, 407, 16, 4777-4782; Sripanyakorn S. et al. (2009). British Journal of Nutrition, 102, 6, 825-834). Nevertheless, the suitable concentration, the speciation of the best and cheapest silicon compound, the optimal pH during application, the determination of the principal activity, and the synergies with other compounds is not yet proposed. There is even not a simple test to demonstrate biological activity in plants for a certain silicon composition.
In summary, Si is currently regarded as an essential nutritional element for plants, and there is ample evidence for beneficial effects in micro-organisms, animals and humans. Silicon is particularly beneficial for plants exposed to abiotic and biotic stresses. During the last decade, evidence has come to exist that, besides mono- and di-silicic acid, also silicates and silicate complexes could be sources of bio-available silicon to some extent.
The bottleneck in using silicic acids or silicates is their tendency to quickly precipitate and polymerise, thereby reducing their bio-availability. Silicates are only soluble in strong alkaline conditions. At pH<95 mono-silicic acid quickly polymerizes, resulting in the formation of trimers, tetramers, and bigger linear or cyclic oligomers. At higher concentrations bigger linear molecules of silicic acid are formed which grow further and start to condense into colloidal structures. Further polymerization of these colloids results in the formation of amorphous silica as precipitate or silica gel. Bioavailable silicon as silicic acid in water is only present at a concentration of <3-5 mM.
Soluble silicates are the alkali metal silicates such as sodium and so potassium silicates. They are synthesized from purified silica and highly soluble under alkaline conditions delivering especially mono- and di-silicates. Industrial preparation of silicates results in a concentrated silicate solution which needs to be diluted appropriately. However, these concentrated silicate solution are not very stable, neither are dilutions thereof. Upon dilution, the pH will drop and depending on the silicon concentration and the presence of multivalent metal ions, insoluble silicates are formed. Reaction with multivalent cations, e.g. from water or media, can cause the corresponding metal silicate to precipitate thereby reducing its bio-availability.
One approach for obtaining bio-available silicon is described in International patent application WO 2003/077657. Here, the use of silicic acid, such as orthosilicic acid, at very low pH and moderately alkalinized with basic compounds lacking free hydroxyl groups resulting in pH lower than 2 is described. No silicates are present in the preparation. This is a complete silicic acid approach.
One approach for obtaining bio-available silicon is described in International patent application WO 2001/047807. This patent describes the production of ortho-silicic acid through hydrolysis of a silicate into a solution of pH 0-4 in the presence of a non toxic solvent (liquid). Osmolytes are not mentioned nor needed for the production of the silicic acid solution. This is again a silicic acid approach.
There is thus a clear need in the art to produce bio-available silicon solutions from a cheap bulk material such as silicates, from which high concentrations can easily be obtained, with a high stability, both in concentrated as in diluted form. There is also a need to dilute the concentrated silicon solution with any type of water. Indeed, the stability of alkaline solubilised silicates upon dilution depends on the composition of the medium.